Members of the Hedgehog (Hh) family of signaling molecules mediate many important short- and long-range patterning processes during invertebrate and vertebrate embryonic, fetal, and adult development. In Drosophila melanogaster, a single hedgehog gene regulates segmental and imaginal disc patterning. In contrast, in vertebrates, a hedgehog gene family is involved in the control of proliferation, differentiation, migration, and survival of cells and tissues derived from all three germ layers, including, e.g., left-right asymmetry, CNS development, somites and limb patterning, chondrogenesis, skeletogenesis and spermogenesis.
The vertebrate family of hedgehog genes includes at least four members or paralogs of the single Drosophila hedgehog gene (WO 95/18856 and WO 96/17924). Three of these members, known as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds and mammals. Dhh is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Ihh is involved in bone development during embryogenesis and in bone formation in the adult; and Shh is involved in multiple embryonic and adult cell types derived from all three lineages. Shh is expressed at high levels in the notochoard and Doorplate of developing vertebrate embryos, and directs cell fate in the developing limb, somites and neural tube. In vitro explant assays as well as ectopic expression of Shh in transgenic animals show that Shh plays a key role in neural tube patterning, Echelard et al., (1993), Cell 75: 1417-30 (1993); Ericson et al., Cell 81: 747-56 (1995); Marti et al, Nature 375: 322-25 (1995); Hynes et al., Neuron 19: 15-26 (1997). Hedgehog signaling also plays a role in the development of limbs (Krauss et al., Cell 75: 1431-44 (1993); Laufer et al., Cell 79: 1165-73 (1994); somites (Fan and Tessier-Lavigne, Cell 79: 1175-86 (1994); Johnson et al., Cell 79: 1165-73 (1994), lungs (Bellusci et al., Devel. 124: 53-63 (1997) and skin (Oro et al., Science 276: 817-21 (1997). Likewise, Ihh and Dhh are involved in bone, gut and germinal cell development (Apelqvist et al., Curr. Biol. 7: 801-804 (1997); Bellusci et al., Dev. Suppl. 124: 53-63 (1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al., Development 121: 3163-74 (1995). Specifically, Ihh has been implicated in chrondrocyte development (Vortkamp et al., Science 273: 613-22 (1996)), while Dhh plays a key role in testes development.
Hedgehog signaling occurs through the interaction of hedgehog protein (e.g., in mammals, Shh, Dhh, Ihh, collectively “Hh”) with the hedgehog receptor, patched (Ptch), and the co-receptor Smoothened (Smo). There are two mammalian homologs of Ptch, Ptch-1 and Ptch-2 (“collectively “Ptch”), both of which are 12 transmembrane proteins containing a sterol sensing domain (Motoyama et al., Nature Genetics 18: 104-106 (1998), Carpenter et al., P.N.A.S. (U.S.A) 95(23): 13630-40 (1998). The interaction of Hh with Ptch triggers a signaling cascade that results in the regulation of transcription by zinc-finger transcriptions factors of the Gli family.
The binding of Hh to Ptch releases Smoothened (Smo), a 7 transmembrane G-coupled protein to then activate an intricate intracellular signal-transduction pathway. The activation of Smo then leads to signaling through a multimolecular complex, including Costal2 (Cos2), Fused (Fu) and suppressor of Fused (Su(Fu)), resulting in nuclear transport of the transcription factor Gli. Ho et al., Curr. Opin. Neurobiol. 12:57-63 (2002); Nybakken et al., Curr. Opin. Genet. Dev. 12: 503-511 (2002); i Altaba et al., Nat. Rev. Neurosci. 3: 24-33 (2002). There are three known Gli transcription factors in verebrates: Gli1, Gli2 and Gli3. While Gli1 is a transcriptional activator that is universally induced in Hh-responsive cells, Gli2 and Gli3 can act either as activators or repressors of transcription depending on the cellular context. Absent Hh signaling, Gli3 is processed into a smaller, nuclear transcriptional repressor that lacks the carboxy-terminal domain of full-length Gli3. Upon activation of Smo, Gli3 protein cleavage is prevented, and the full-length form with transcription-activation function is generated. Gli2 also encodes a repressor function in its carboxy-terminally truncated form, but its formation does not appear to be regulated by Hh signaling. Stecca et al, J. Biol. 1(2):9 (2002).
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)). Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
Hedgehog signaling has been implicated in a wide variety of cancers and carcinogenesis. One example of the carcinogenic process is vascularization. Angiogenesis, the process of sprouting new blood vessels from existing vasculature and arteriogenesis, the remodeling of small vessels into larger conduct vessels are both physiologically important aspects of vascular growth in adult tissues (Klagsbrun and D'Amore, Annu. Rev. Physiol. 53: 217-39 (1991); Folkman and Shing, J. Biol. Chem. 267(16): 10931-4 (1992); Beck and D'Amore, FASEB J. 11(5): 365-73 (1997); Yancopoulos et al., Cell 93(5): 661-4 (1998); Buschman and Scaper, J. Pathol. 190(3): 338-42 (2000). These processes of vascular growth are also required for beneficial processes such as tissue repair, wound healing, recovery from tissue ischemia and menstrual cycling. However, they are also required for the development of pathological conditions such as the growth of neoplasias, diabetic retinopathy, rheumatoid arthritis, psoriasis, certain forms of macular degeneration, and certain inflammatory pathologies (Cherrington et al., Adv. Cancer Res. 79:1-38 (2000). Thus, the inhibition of vascular growth can inhibit cellular proliferation, growth, differentiation and/or survival. As Hh has been shown to promote angiogenesis, Hh antagonists would be expected to possess anti-angiogenic properties.
The gene BOC [brother of CDO or regional cell adhesion molecule-related/down-regulated by oncogenes (Cdon) binding protein] encodes a type I plasma membrane protein having an Ig/FNIII repeating domain, and which likely functions as a receptor subunit for cell-cell communications. BOC protein is known to interact with CDO (cell adhesion molecule-related/down-regulated by oncogenes), N-cadherins, and M-cadherins in a cis fashion, forming a receptor complex at sites of cell-cell contact in myoblasts. Kang et al., PNAS 100(7): 3989-3994 (2003).
Like BOC, CDO is also a type I cell surface receptor protein, further sharing similar similar ectodomain (EC+TM domain) structural features, such as Ig repeats and Fibronectin (FN) type III repeats. More precisely, as shown in FIG. 6, BOC has five Ig repeats, and 3 FN repeats, while CDO has four Ig repeats and three FNIII repeats. However, the intracellular domains of BOC and CDO do not share significant homology. SiRNA knockdown of CDO in Drosophila leads to loss of hedgehog signaling responses. Lum et al., Science 299: 2039-2044 (2003). Others have shown that mutation of CDO in mammals results in a microform of holoprosencephaly (HPE), which is suggestive of involvement in hedgehog signaling. Cole et al., Curr. Biol. 13: 411-415 (2003). However, while HPE is a phenotype of hedgehog signaling failure, Cole et al. also points out that less than 15% of all cases of naturally occurring HPE result from mutations in hedgehog signaling components. Thus, HPE alone is not definitive of involvement in hedgehog signaling.
During embryonic development, BOC and CDO are expressed in the musculoskeletal and central nervous systems and in areas of proliferation and differentiation. BOC and CDO has further been associated with myogenic differentiation (Kang et al, EMBO J. 21(1&2): 114-124 (2002) and macrophage defects (PCT/US2006/019651, filed 18 May 2006). Expression of CDO and BOC in myoblast cell lines is downregulated by the ras oncogene, and forced re-expression of either CDO or BOC can override ras-induced inhibition of myogenic differentiation. Kang et al., J. Cell Biol. 143:403-413 (1998); Kang et al., EMBO J. 21:114-124 (2002). The promyogenic properties of CDO and BOC were further shown to be present in the human rhabdomyosarcoma cell line, RD. Stable overexpression of CDO or BOC in RD cells led to enhanced expression of two markers of muscle cell differentiation, troponin T and myosin heavy chain, and to increased formation of elongated, myosin heavy chain-positive myotubes. It has further been suggested that CDO and BOC play a role in the inverse relationship between differentiation and transformation of cells in the skeletal muscle lineage. Wegorzewska et al., Mol. Carcinogenesis. 37(1): 1-4 (2003).
Applicants demonstrate herein that both BOC and CDO can bind to Shh and differentially regulate hedgehog signaling, operating in tandem through a negative feedback mechanism. While BOC overexpression can inhibit Shh signaling to a level similar as Ptch1 overexpression, CDO Δ(cyt) overexpression (CDO lacking the cytoplasmic domain) potentiated Hh signaling at suboptimal Shh concentrations. This suggests that BOC can sequester or antagonize Hh signaling, while CDO can amplify or agonize Hh signaling. Moreover, BOC and CDO, as well as antagonists thereof, could be an effective therapeutic to treat disorders that are implicated by aberrent hedgehog signaling.